The present invention relates generally to concurrent flow mixing methods and apparatuses that are adapted for preparation of gene therapeutics, as well as compositions prepared thereby. In the various embodiments, the present invention provides controlled and uniform mixing of gene therapy vectors and gene therapy vector vehicles for improved reproducibility, scaleability, stability, and pharmaceutical efficacy.
Ideally, techniques for nucleic acid delivery include one or more attributes, such as, for example: 1) efficient preparation of the nucleic acid delivery composition, including simplified operation, cost effectiveness, consistency, stability and uniformity; 2) efficient delivery and incorporation of the nucleic acid into the host cell; 3) avoidance of undesirable side effects such as cell toxicity, or the introduction of unwanted elements (e.g., additional viral genes); and 4) selective targeting of the nucleic acid to the desired host cell.
Various methods have been employed to introduce foreign genes into cells. Since DNA is a large and bulky molecule, it is no simple matter to introduce such molecules into cells, not only due to the size of the molecule but due to its chemical and charge-related characteristics. Therefore, many gene therapy methods endeavor to find a way to xe2x80x9cpackagexe2x80x9d the therapeutic DNA in such a manner that it may be transported across the membranes of the target cells. As those of skill in the art are well aware, this is not an easily-achieved goal. Moreover, most xe2x80x9cpackagesxe2x80x9d are comprised of components which interact with each other based on cooperative kinetics. Such kinetics are associated with bringing together components that each contain a variety of cooperative binding sites, thereby adding a level of complexity which when poorly controlled may lead to non-uniform compositions. Accordingly, to date, no methods are known to have been developed which control the cooperative kinetics involved in the manufacture of gene therapeutic compositions nor have methods been developed which allow for consistent, reproducible, and uniform (i.e., substantially homogenous) gene therapeutic compositions to be made.
One method of introducing therapeutic nucleic acids into host cells utilizes polycation/nucleic acid condensates. See, for example U.S. Pat. Nos. 5,166,320 and 5,635,383 as well as published International Patent App. Nos. WO 93/04701 and WO 94/06922. This methodology uses polycations to condense the nucleic acid into a compact structure so as to facilitate cellular uptake. However, such methodologies suffer from poorly controlled conditions for forming the condensates, thereby leading to aggregation or poorly condensed compositions.
Another current method of introducing foreign (or therapeutic) DNA into cells uses precipitation of DNA with calcium phosphate to form insoluble particles. Ideally, these particles become internalized in the host cells (via endocytosis) and induce expression of the new gene. Internalization of such particles, however, is independent of an endocytosis recognition site, so the internalization is non-specific and is thus not targetable to particular cells and organs. While generally applicable to in vitro applications, this process has more limited applicability in in vivo applications due to the insolubility of the DNA co-precipitate particles and the inability to control uptake and targeting specificity. Additionally, conditions necessary for internalization of DNA using the calcium phosphate method may be harsh and cause cell death.
An alternate method of introducing foreign genes into cells utilizes liposomes. Liposome-encapsulated DNA has been utilized both in vitro and in vivo. This technique, however, suffers from difficulties in controlling liposome size, which inherently affects uniform and controlled delivery to the target cell. There are also problems associated with keeping the liposome intact throughout the cell delivery process and difficulties associated with achieving specific targeting.
Other methods of delivering exogenous nucleic acids to cells have been proposed for use but suffer from a variety of limitations that make them impractical for gene therapy applications. For example, electroporation is impractical as the conditions required for gene delivery are harsh and lead to toxicity and cell death. Similarly, injection of naked DNA is unpredictable, especially with respect to targeting specificity, controlled uptake, and immunogenicity. Microinjection of nucleic acids into cells is very labor intensive, because each cell must be individually manipulated, and thus it is not a practical alternative to bolus injections of DNA, although it avoids some of the problems associated with bolus injections. Thus, it is clear that none of the foregoing techniques provide a practical alternative for in vivo therapeutic applications.
The preparation of gene therapy vectors according to the methods described herein thus provides attractive means to improve upon known techniques and develop new gene transfer methods. The preparation and use of such vectors is not without its own unique limitations, however, as many have observed. Fortunately, the methods and apparatuses of the present invention overcome many of the difficulties experienced by others and enhance the efficacy of gene therapy for pharmaceutical commercialization.
Various methods of compacting nucleic acids to facilitate their entry into cells are described in the art and are useful to underscore the novel aspects of some embodiments of the present invention. The following examples are provided to illustrate some of those methods.
International Patent Publication WO 95/25809 describes nucleic acids that are compacted to facilitate their uptake by target cells of an organism. The publication further describes methods for compacting nucleic acids and therapeutic uses of the compacted DNA for delivery across the membrane of living cells.
International Patent Publication WO 96/41606 describes synthetic virus-like particles containing a plurality of peptides capable of condensing nucleic acid and condensed nucleic acid. The synthetic virus-like particle is self-assembling and may be designed to deliver nucleic acid to be incorporated into the chromosomal or extrachromosomal sequences of target cells. These particles are described as being useful for transfecting mammalian cells.
Other systems that have been developed to deliver nucleic acid molecules to mammalian cells include, emulsions (e.g., liposomes), nanoparticles, microparticles, and similar heterogeneous systems. However, these systems also suffer from lack of scaleable production methodologies that produce uniform, reproducible and highly efficacious compositions.
Of the aforementioned methods, compaction or condensation of the nucleic acids for delivery into the host cell offers great potential. For this technique to be fully exploited, however, methods and apparatuses for producing suitable nucleic acid complexes on a variety of scales, including for example, laboratory scale, commercial production scale, and individual patient bedside administration scale, are desirable. While a variety of mixing devices are available, some of which are exemplified below, they are not generally applicable to gene therapy vector and vehicle compositions and especially with regard to nucleic acid compaction or condensation methods.
For example, U.S. Pat. No. 4,908,187 describes a diluting and mixing device which is capable of diluting a first solution to produce a second solution which is mixed with an undiluted third solution to produce a unique series of combined solutions. Such a device is intended for use in obtaining kinetic analysis (e.g., chemical, biochemical or physical chemical) data on reactions in solution.
U.S. Pat. No. 4,979,942 describes a two-component syringe delivery system. In this system, two reactive fluids are delivered simultaneously and separately from a pair of syringes to a delivery site. The tubing exiting one syringe passes through a cannula exiting from the other syringe to deliver both fluids separately, but in a controlled volume and space to a delivery site. Alternatively, the fluids can mix within the cannula; purportedly, its configuration prevents clogging at the delivery site.
U.S. Pat. Nos. 4,978,336 and 5,116,315 describe a biological syringe system for delivering a first and second fluid in a mixed composition comprising a manifold and a discharge assembly. The discharge assembly has a mixing space and further comprises a mixing mechanism to thoroughly mix the first and second fluids and to immediately thereafter atomize the thoroughly mixed fluids in a spray discharge from the discharge assembly. This apparatus is described as being particularly useful for applying a chemically formed tissue adhesive and protective covering.
U.S. Pat. No. 5,505,704 describes a hand-held liquid medication injector having dual, bidirectional dosage metering mechanisms for permitting a variable dosage amount for each cartridge of liquid medication. The medication injector has an injection mechanism independent of the metering mechanism that loads and injects the liquid medication.
The apparatuses described above are similar to each other in that they are used to mix two solutions. There is no indication, however, that these apparatuses would be useful or suitable for providing uniform gene therapy vector mixtures or uniform condensate particles, particularly where the control of such uniformity and/or condensate particle size is critical. Accordingly, the need exists for methods and apparatuses to make gene therapeutic vector and vehicle mixtures of uniform quality, of a defined particle size in a convenient manner (e.g., when using a condensing agent), and ideally in a manner that is adaptable enough to be amenable to scale-up without sacrificing the quality of the compositions produced.
The invention relates to methods and apparatuses, including xe2x80x9cconcurrent flow mixingxe2x80x9d methods and apparatuses, for providing gene therapy vector and vehicle compositions (e.g., mixtures, complexes, and condensates) of precisely controlled uniformity (i.e., substantially homogenous) as well precisely controlled particle size for condensate complexes. These methods and apparatuses simultaneously control the rate of introduction of reactants, mixing of the reactants and removal of the resulting mixture. Accordingly, the methods and apparatuses of the present invention are capable of regulating the cooperative kinetics of the formation of various gene therapy vector and vehicle mixtures, complexes, and condensates (collectively, compositions).
In one embodiment, methods are provided for the preparation of gene therapy compositions, comprising concurrently introducing at least a first molecular entity-containing solution and a second molecular entity-containing solution, each in a controlled and independent manner, into at least a first flow-through mixer such that the two solutions contact, mix, and form a uniform mixture, such that said uniform mixture exits from the flow-through mixer at a controlled rate, and wherein said first and second molecular entity-containing solutions collectively comprise at least one gene therapy vector and at least one gene therapy vehicle.
In the various embodiments, described herein, the mixer may be a static mixer or may be a dynamic mixer.
In another aspect, the invention provides a method of preparing a gene therapy composition, comprising DNA:condensing agent condensation complex of a predetermined size comprising the steps of: a) providing i) a DNA-containing solution, ii) a condensing agent-containing solution, and iii) a flow-through mixer; and b) concurrently introducing said DNA-containing solution and said condensing agent-containing solution, each in a controlled and independent manner, into said mixer such that the two solutions contact, mix, and form a mixed solution containing a DNA:condensing agent condensation complex of predetermined size thereby, and such that said condensation complex exits from the flow-through mixer at a controlled rate such that further reaction of the desired DNA:condensing agent condensation complex with additional DNA- and/or condensing agent-containing solution is minimized or avoided.
The methods and apparatuses described herein include those wherein the condensing agent comprises a polycationic molecule. Polycationic molecules useful in the methods and apparatuses of the invention are exemplified by molecules including, for example, polycationic peptides or polypeptides; polycationic proteins; polycationic polyamino acids; polycationic carbohydrates; polycationic synthetic polymers; polycationic small synthetic organic amines; inorganic multivalent cations; cationic lipids; and synthetic viral particles.
The methods and apparatuses described herein are suitable for providing molecular entity condensation complexes of uniform particle size, including, for example, complexes having a particle size of about 2000 nm or smaller, alternatively of about 1000 nm or smaller, alternatively of about 500 nm or smaller, alternatively of about 200 nm or smaller, alternatively of about 100 nm or smaller, or alternatively of about 50 nm or smaller. The methods described herein are also suitable for providing condensation complexes in a range of particle sizes, including for example, from about 30 to about 2000 nm, alternatively from about 30 to about 200 nm, or alternatively of from about 30 to about 100 nm.
In another embodiment, the invention relates to apparatuses for preparing gene therapy compositions comprising: a) a first molecular entity-containing solution dispenser; b) a second molecular entity-containing solution dispenser; c) a mixer attached to a solution removal outlet, wherein the first molecular entity-containing solution dispenser and the second molecular entity-containing solution dispenser are each independently connected to the mixer; and d) a solution flow controller that independently controls the rate of introduction of the first molecular entity-containing solution and the second molecular entity-containing solution into the mixer, and the rate of flow of the resulting mixed solution through the mixer and out to the solution removal outlet, and wherein said first and second molecular entity-containing solutions collectively comprise at least one gene therapy vector and at least one gene therapy vehicle. Such a method is referred to herein as concurrent flow mixing (CFM).
In further embodiments, the invention relates to apparatuses for preparing gene therapy compositions, comprising: a) a first molecular entity-containing solution introduction means; b) a second molecular entity-containing solution introduction means; c) a mixing means attached to a solution removal means, wherein the first molecular entity-containing solution introduction means and the second molecular entity-containing solution means are each independently connected to the mixing means; and d) a solution flow controller that independently controls the rate of introduction of the first molecular entity-containing solution and the second molecular entity-containing solution into the mixing means, and the rate of flow of the resulting mixed solution through the mixing means and out to the solution removal means.
In other embodiments, the invention provides an apparatus for preparing a DNA:condensing agent condensation complex of a predetermined size comprising: a) a DNA-containing solution dispenser; b) a condensing agent-containing solution dispenser; c) a mixer attached to a solution removal outlet, wherein the DNA-containing solution dispenser and the condensing agent-containing solution dispenser are each independently connected to the mixer; and d) a solution flow controller that independently controls the rate of introduction of the DNA-containing solution and the condensing agent-containing solution into the mixer, the size of the DNA:condensing agent condensation complex formed thereby, and the rate of flow of the resulting mixed solution through the mixer and out to the solution removal outlet.
In even further embodiments, the invention provides an apparatus, comprising: a) a DNA-containing solution introduction means; b) a condensing agent-containing solution introduction means, c) a mixing means attached to a solution removal means, wherein the DNA-containing solution introduction means and the condensing agent-containing solution means are each independently connected to the mixing means, and d) a solution flow controller that independently controls the rate of introduction of the DNA-containing solution and the condensing agent-containing solution into the mixing means, the size of the DNA:condensing agent condensation complex formed thereby, and the rate of flow of the resulting mixed solution through the mixing means and out to the solution removal means.
In addition, the methods and apparatuses of the present invention may readily be modified to accommodate xe2x80x9cstackingxe2x80x9d or xe2x80x9cpiggybackingxe2x80x9d of components (e.g., dispensers, flow controllers, etc.). For example, FIG. 2 illustrates another embodiment of such an apparatus.
One example of a rationale for xe2x80x9cpiggybackingxe2x80x9d would be to allow multiple reactant solutions to be admixed. Another example would be to allow multiple components to be connected to a single flow controller. Other rearrangements, interconnections and modifications are contemplated and are within the scope of this invention, as well as within the purview of the skilled artisan to accomplish, following the teachings of this application.
Thus, the present invention also discloses methods and apparatuses that accommodate xe2x80x9cbranchedxe2x80x9d configurations, whereby multiple components (e.g., dispensers, mixers, flow controllers, etc.) may be used in a single system. For example, one nucleic acid may be admixed with a first condensing agent, and a second nucleic acid may be admixed with a second condensing agent, and then the two complexes may thereafter be admixed to form a condensation complex comprising two different nucleic acids, two different condensing agents, or combinations of same. In another example, a nucleic acid (e.g., DNA) may be admixed with a first condensing agent, ligand, other reagent, or some combination of the same to form a first condensation complex; subsequently, that first condensation complex may be admixed with a second condensing agent, ligand, other reagent, or some combination of the same to form a second condensation complex. A wide variety of condensation complexesxe2x80x94particularly those having increased stability and/or particle size uniformityxe2x80x94may be made using the methods and apparatuses of the present invention and are thus contemplated within the scope of the present invention, as well.
Other embodiments of the invention are disclosed herein and include methods and apparatuses wherein the first molecular entity is a nucleic acid and the second molecular entity is a condensing agent. In various disclosed embodiments, the nucleic acid is a therapeutic nucleic acid; in other embodiments, the nucleic acid or therapeutic nucleic acid is DNA.
In further embodiments, compositions produced by the methods and apparatuses, described herein, are provided.